REPORTS

GOS scaffolds, finding 46 MpnS and 20 HepD homologs, using a protein basic local alignment search tool (BLASTP) cutoff value of 1010 (table S2). No HppE homologs were observed. None of the HepD homologs were identified when N. maritimus MpnS was used as the query sequence; likewise, none of the MpnS homologs were identified when HepD was used as a query. Thus, BLASTP clearly differentiates between the two homologous groups, supporting the assignment of the recovered sequences as MpnS and HepD proteins, respectively. To independently support these functional assignments, we constructed maximumlikelihood phylogenetic trees including biochemically validated MpnS, HepD, and HppE proteins (Fig. 3A and fig. S9). We also used a hierarchical clustering method to examine all putative and validated MpnS, HepD, and HppE proteins (fig. S10). In both cases, robust support for the functional assignments was obtained. Thus, we conclude that the recovered GOS MpnS homologs are likely to be methylphosphonate synthases. Additional support for the function of the MpnS homologs was revealed by analysis of neighboring genes found in GOS DNA scaffolds (Fig. 3B and table S3). Many of the nearby open reading frames are homologous to those found in the N. maritimus gene cluster, including the phosphonate biosynthetic genes ppm, ppd, and pdh, as well as homologs of the sulfatases and nucleotidyl transferase genes, suggesting that the GOS scaffolds encode genes for the synthesis of similar MPn esters. Several other genes found on the scaffolds provide evidence for the identity of the organisms in which they are found. One of the scaffolds includes a 23S ribosomal RNA gene that can be confidently placed within the SAR11 clade between Pelagibacter species (fig. S11), whereas two of the manC genes are nearly identical to ones found in Pelagibacter sp. HTCC7211. Although the mpnS gene is absent in sequenced Pelagibacter genomes, these data strongly support the conclusion that some members of this genus have the capacity to synthesize MPn. Relatives of Nitrosopumilus and Pelagibacter are among the most abundant organisms in the sea, with global populations estimated at 1028 for both ammonia-oxidizing Thaumarchaeota (14) and members of the SAR11 clade (22). Thus, the observation of mpnS in some members of these genera is consistent with the idea that MPn synthesis is prevalent in marine systems. To provide direct support for this notion, we measured the abundance of the mpnS gene relative to the abundance of typical single-copy genes as previously described (23). We also quantified the occurrence of the ppm gene to provide an estimate of the relative occurrence of phosphonate synthesis in general (table S4). Based on these data, we estimate that ~16% of marine microbes are capable of phosphonate biosynthesis, whereas 0.6% have the capacity to synthesize MPn. Because the GOS samples are confined to the upper few meters of the ocean, extrapolation of this analysis to the deeper ocean should be viewed with some skepticism. Nevertheless, the upper 200 m of the worlds oceans are thought to contain ~3.6 1028 microbial cells, with an average generation time of ~2 weeks (24). Thus, even with the relatively modest abundance of MPn biosynthesis suggested by our data, it seems quite possible that these cells could provide sufficient amounts of MPn precursor to account for the observed methane production in the aerobic ocean via the C-P lyasedependent scenario suggested by Karl et al. (2).
References and Notes
1. J. E. Rogers, W. B. Whitman, Eds., Microbial Production and Consumption of Greenhouse Gases: Methane, Nitrogen Oxides, and Halomethanes (American Society for Microbiology, Washington, DC, 1991). 2. D. M. Karl et al., Nat. Geosci. 1, 473 (2008). 3. W. S. Reeburgh, Chem. Rev. 107, 486 (2007). 4. C. G. Daughton, A. M. Cook, M. Alexander, FEMS Microbiol. Lett. 5, 91 (1979). 5. A. Martinez, G. W. Tyson, E. F. Delong, Environ. Microbiol. 12, 222 (2010). 6. I. N. Ilikchyan, R. M. L. McKay, J. P. Zehr, S. T. Dyhrman, G. S. Bullerjahn, Environ. Microbiol. 11, 1314 (2009). 7. L. L. Clark, E. D. Ingall, R. Benner, Am. J. Sci. 299, 724 (1999). 8. L. L. Clark, E. D. Ingall, R. Benner, Nature 393, 426 (1998). 9. W. W. Metcalf, W. A. van der Donk, Annu. Rev. Biochem. 78, 65 (2009). 10. S. A. Borisova, B. T. Circello, J. K. Zhang, W. A. van der Donk, W. W. Metcalf, Chem. Biol. 17, 28 (2010). 11. J. A. Blodgett, J. K. Zhang, W. W. Metcalf, Antimicrob. Agents and Ch. 49, 230 (2005). 12. A. C. Eliot et al., Chem. Biol. 15, 765 (2008). 13. B. T. Circello, A. C. Eliot, J. H. Lee, W. A. van der Donk, W. W. Metcalf, Chem. Biol. 17, 402 (2010). 14. M. B. Karner, E. F. DeLong, D. M. Karl, Nature 409, 507 (2001). 15. M. Knneke et al., Nature 437, 543 (2005). 16. Z. Shao et al., J. Biol. Chem. 283, 23161 (2008). 17. H. M. Seidel, S. Freeman, H. Seto, J. R. Knowles, Nature 335, 457 (1988). 18. Materials and methods are available as supplementary materials on Science Online. 19. J. C. Tebby, Ed., CRC Handbook of Phosphorus-31 Nuclear Magnetic Resonance Data (CRC Press, Boca Raton, FL, 1991). 20. R. L. Hilderbrand, Ed., The Role of Phosphonates in Living Systems (CRC Press, Boca Raton, FL, 1983). 21. S. Yooseph et al., PLoS Biol. 5, e16 (2007). 22. R. M. Morris et al., Nature 420, 806 (2002). 23. E. C. Howard, S. Sun, E. J. Biers, M. A. Moran, Environ. Microbiol. 10, 2397 (2008). 24. W. B. Whitman, D. C. Coleman, W. J. Wiebe, Proc. Natl. Acad. Sci. U.S.A. 95, 6578 (1998). Acknowledgments: This work was supported by the NIH (grants GM PO1 GM077596 and F32 GM095024), the Howard Hughes Medical Institute (HHMI), and NSF (grants MCB-0604448, OCE-1046017, and MCB-0920741). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of General Medical Sciences, NIH, NSF, or HHMI. The authors thank L. Zhu (University of Illinois at UrbanaChampaign) for valuable help with NMR experiments.

Supplementary Materials
www.sciencemag.org/cgi/content/full/337/6098/1104/DC1 Materials and Methods Figs. S1 to S11 Tables S1 to S4 References (2539) 31 January 2012; accepted 10 July 2012 10.1126/science.1219875

The Shared Antibiotic Resistome of Soil Bacteria and Human Pathogens

Kevin J. Forsberg,1* Alejandro Reyes,1* Bin Wang,1,2 Elizabeth M. Selleck,3 Morten O. A. Sommer,4,5 Gautam Dantas1,2 Soil microbiota represent one of the ancient evolutionary origins of antibiotic resistance and have been proposed as a reservoir of resistance genes available for exchange with clinical pathogens. Using a high-throughput functional metagenomic approach in conjunction with a pipeline for the de novo assembly of short-read sequence data from functional selections (termed PARFuMS), we provide evidence for recent exchange of antibiotic resistance genes between environmental bacteria and clinical pathogens. We describe multidrug-resistant soil bacteria containing resistance cassettes against five classes of antibiotics (b-lactams, aminoglycosides, amphenicols, sulfonamides, and tetracyclines) that have perfect nucleotide identity to genes from diverse human pathogens. This identity encompasses noncoding regions as well as multiple mobilization sequences, offering not only evidence of lateral exchange but also a mechanism by which antibiotic resistance disseminates. he continued evolution and widespread dissemination of antibiotic resistance genes in human pathogens is a preeminent clinical challenge (1). Environmental reservoirs have long been implicated as a source of resistance found in human pathogens (2). However, apart from certain opportunistic bacterial pathogens, among which the same species can be found in the environment or infecting humans (3), examples of resistance genes from environmental bacteria with high identity to those of pathogens

are rare (4, 5). The two documented examples are of Kluyvera and Shewanella isolates, which are found free-living in environmental settings (5, 6) yet have resistance genes (CTX-M b-lactamase and qnrA genes, respectively) with high identity (100% identity in clinical Kluyvera isolates) to those of pathogens (4, 5). The limited examples of resistance genes shared between environmental microbes and human pathogens raise questions regarding the clinical impact of environmental resistance. For instance, whether shared resistance

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